The polo-like kinase 1 (Plk1) is considered to be an attractive anticancer molecular target, due to its central role in cell division. Plk1 is overexpressed in a wide spectrum of human cancers and its overexpression is thought to promote genomic instability and tumorigenesis. Upregulated Plk1 activity also appears to be closely associated with the aggressiveness and poor prognosis of these cancers. Additionally, various cancer cells, but not their isogenic normal cells, can become addicted to Plk1 overexpression for their viability and this can exacerbate the vulnerability of these cells to anti-Plk1 drugs. Targeting Plk1 may permit induction of cancer cell-selective mitotic block and apoptotic cell death in Plk1-addicted cancers. A potential drawback of classical inhibitors directed at the Plk1 kinase domain (KD) may arise from a lack of specificity due to the high degree of similarity in the ATP binding clefts among kinases. For example, the Plk1 kinase inhibitor, volasertib (BI6727), has faltered in Phase III clinical trials, presumably because it inhibits Plk2 and Plk3 (contraindicated targets) with IC50 values that are similar to Plk1. Improving Plk1 specificity is one of the most pressing concerns to address to accomplish better clinical outcomes with less toxicological problems. Plk1 also consists of a non-catalytic polo-box domain (PBD), which binds to the enzyme's physiological substrates and localizes the enzyme to discrete locations within the kinetochore. However, Plk1 does not engage each substrate individually and not all of its substrates require a prior interaction with the PBD before being phosphorylated by its KD. This suggests that targeting the PBD may serve as a target-restricted strategy for developing anti-Plk1 therapeutics, which may antagonize a subset of Plk1 functions dysregulated in cancer. Importantly, unlike ATP-competitive inhibitors, whose specificities must be obtained against more than 500 other cellular kinases, PBD inhibitors target a structurally unique domain found in only four proteins (Plk1-3 and Plk5). Additionally, while ATP-competitive inhibitors can abrogate all Plk1-dependent biochemical processes indiscriminately in both cancer and normal cells, PBD inhibitors interfere only with PBD-dependent Plk1 functions. It is possible that anti-PBD agents could potentially be optimized so that they selectively inhibit a subset of PBD-dependent interactions, which are enriched in biochemically rewired, Plk1-addicted cancer cells. Inhibition of Plk1 PBD function alone is sufficient for effectively imposing mitotic arrest and apoptotic cell death in cancer cells but not in normal cells. The Plk1 PBD functions by recognizing and binding to to phosphothreonine (pT)/phosphoserine (pS)-containing protein sequences. We are engaged in efforts to develop Plk1 PBD-binding inhibitors. Starting from the 5-mer phosphopeptide PLHSpT, we recently identified peptidic inhibitors that showed from 1000- to more than 10,000-fold improved PBD-binding affinity. In collaboration with Dr. Michael Yaffe (MIT), X-ray co-crystal structures of these peptides bound to Plk1 PBD indicated unanticipated modes of binding that take advantage of a cryptic binding channel that is not present in the non-liganded PBD or engaged by the parent pentamer phosphopeptide. The cryptic pocket is accessed by means of a -(CH2)8Ph moiety attached to the N(pi) nitrogen of the His imidazole ring (which we desigate as His* or H*). Although critical elements in the high affinity recognition of peptides and proteins by PBD are derived from pT/pS-residues, the use of these residues in therapeutics is potentially limited by poor cellular uptake, in part due to high anionic charge of the phosphoryl moiety. We have recently discovered new synthetic transformations that introduces alkyl groups onto the N(tau) nitrogen of the His* residues, which reduce the overall peptide anionic charge by intramolecular charge masking. This provides peptides with enhanced efficacy in cellular assays. We have further modified these peptides by introducing bio-reversible prodrug protection of one phosphoryl acidic hydroxyl. This yielded neutral peptides that show even greater cellular efficacies. We developed an efficient synthesis of phosphonomethyl phenylalanine (Pmab), a phosphatase-stable analog of phosphothreonine (pThr). We used this protocol to prepare Pmab variants having different substituents at the 3R-center. When incorporated into our peptidomimetic scaffold, certain of these new Pmab analogs exhibit Plk1 PBD-binding affinities, which are several-fold higher than Pmab. We realized that access to bis-N(pi), N(tau)-derivatized His residues opens unexplored access to new families of peptidomimetics, particularly new classes of macrocycles. Macrocyclization is a classical approach for conformational restriction and ring closure using a bifurcated His residue, which could provide novel opportunities for novel structural variation. Co-crystal structures of Plk1 PBD-bound open-chain peptide PLH*SpT show that the His* imidazole N(tau)-nitrogen is approximately 5-6 angstroms from the C-terminus. We found that microwave-assisted on-resin alkylation of N(tau)-nitrogens of His*-containing peptides proceeds efficiently with alkyl iodide electrophiles. In this manner, we installed Boc-protected alkyl amines of increasing length methylene linkers at the N(tau)-position on-resin. Following cleavage from the resin, the resulting peptides were subjected to solution-phase macrocyclization by amide formation of the N(tau)-tethered amine with a C-terminal carboxyl group to yield cyclic constructs containing 4 - 6 methylene linkers. Certain of these constructs demonstrated up to 3-fold improved PBD-binding potency. An X-ray co-crystal structure of the highest affinity ligand demonstrated near perfect overlay with the open chain parent peptide. Importantly, deleting the N-terminal Pro-Leu moiety from the pentapeptide macrocycle provided a cyclic His-Ser-pThr tripeptide with Plk1 PBD-binding potency similar to the open chain PLH*SpT, while having improved Plk1 selectivity. Relative to the open-chain parent, this represents a charge-masked cyclic tripeptide with smaller molecular weight yet equal affinity. We are actively pursuing this construct as a lead toward PBD-targeted peptidomimetics with improved cell-membrane permeability and cell-based activity.